9-Ethynyl noscapine induces G2/M arrest and apoptosis by disrupting tubulin polymerization in cervical cancer.

Noscapine is a phthalide isoquinoline alkaloid present in the latex of Papaver somniferum and has demonstrated potent antitumor activity in various cancer models. Structural changes in the core molecule of noscapine architecture have produced a number of potent analogs. We have recently synthesized the novel noscapine analogs (3, 4, and 5) with different functional groups appended at ninth position of natural noscapine. The anticancer activity of these compounds has been investigated using various human cancer cell lines such as HeLa (cervical cancer), DU-145 (prostate cancer), MCF-7 (breast cancer), and IMR-32 (neuroblastoma). One of the compounds in this series, 9-ethynyl noscapine (5), has demonstrated good anticancer activity against HeLa cells. Biological studies demonstrated that compound 5 decreased cell viability and colony formation in HeLa cells in a concentration dependent manner. To further uncover the mechanism in detail, we evaluated compound 5 effect on cell cycle progression, microtubule dynamics, and apoptosis. Cell cycle and western blotting analysis revealed that 9-ethynyl noscapine treatment resulted in cell cycle arrest at G2/M and decreased CDK1 and cyclinB1 protein expression. We also observed that 9-ethynyl noscapine (5) treatment leads to disruption in tubulin polymerization and induction of apoptosis by decreasing expression of bcl2, pro-caspase 3, and activation of cytochrome C. Taken together, our results indicate that 9-ethynyl noscapine (5) effectively supresses the growth of cervical cancer cells (HeLa) by disrupting tubulin polymerization, cell cycle progression leading to apoptosis.

Quisinostat mediated autophagy is associated with differentiation in neuroblastoma SK-N-SH cells.

Neuroblastoma (NB) is the most common childhood cancer that arises from the sympathetic nervous system. NB is characterized by poor prognosis. One of the strategies to control NB is activating the differentiation process in undifferentiated NB cells. Many differentiating agents including 13-cis-retinoic acid (RA) led to disappointing results. In the current study, we investigated the effect of Quisinostat/JNJ-26481585(JNJ) on NB SK-N-SH cells differentiation. The SK-N-SH cell differentiation was observed by morphology and neurite length measurement. The cell cycle arrest was determined by FACS analysis. The relative levels of autophagy marker LC3-II, neuronal markers ßIII-tubulin and Eno-2, cell cycle related proteins cyclin D1 and CDK 4 were detected by western blotting. JNJ induces differentiation in SK-N-SH cells, as evident by the morphological features and expression of neuronal markers, ßIII-tubulin and Eno-2. Cell cycle arrest at G1 phase was confirmed by a decrease in the expression of cyclin D1 and CDK 4. Furthermore, we also observed that autophagy plays an important role in JNJ induced cell differentiation of SK-N-SH cells. We demonstrated that autophagy is induced upon JNJ treatment and is important for the neuronal differentiation of human SK-N-SH cells.

Inhibition of JNJ-26481585-mediated autophagy induces apoptosis via ROS activation and mitochondrial membrane potential disruption in neuroblastoma cells.

Neuroblastoma (NB) is the common pediatric tumor of the sympathetic nervous system characterized by poor prognosis. Owing to the challenges such as high tumor heterogeneity, multidrug resistance, minimal residual disease, etc., there is an immediate need for exploring new therapeutic strategies and effective treatments for NB. Herein, in the current study, we explored the unexplored response of NB cells to the second-generation histone deacetylase inhibitor (HDACi) JNJ-26481585(JNJ) and the lysosomotropic agent, Chloroquine (CQ) alone and upon JNJ/CQ treatment as a plausible therapeutic. We identify that while JNJ alone induced autophagy in NB cells, JNJ/CQ treatment decreased the viability and proliferation of NB cells in vitro by switching from autophagy to apoptosis. Further we found that autophagy inhibition by CQ pre-treatment led to the generation of ROS and a decrease in the mitochondrial membrane potential (MMP) that subsequently caused caspase-3-mediated apoptotic cell death in NB cells. Corroborating the above observations, we found that the ROS scavenger N-acetylcysteine (NAC) countered caspase-3 activity and the cells were rescued from apoptosis. Finally, these observations establish that JNJ/CQ treatment resulted in cell death in NB cells by triggering the formation of ROS and disruption of MMP, suggesting that modulation of JNJ-induced autophagy by CQ represents a promising new therapeutic approach in NB.

Enniatin A Analogues as Novel Hsp90 Inhibitors that Modulate Triple-Negative Breast Cancer.

The 90 kilo-Dalton heat shock protein (Hsp90) is a molecular chaperone that facilitates the maturation of nascent polypeptides into their biologically active conformation. Because many of the >400 known client protein substrates are implicated in the development/progression of cancer, it is hypothesized that Hsp90 inhibition will simultaneously shut down numerous oncogenic pathways. Unfortunately, most of the small molecule Hsp90 inhibitors that have undergone clinical evaluation thus far have failed due to various toxicities. Therefore, the disruption of Hsp90 protein-protein interactions with cochaperones and/or client substrates has been proposed as an alternative way to achieve Hsp90 inhibition without such adverse events. The hexadepsipeptide Enniatin A (EnnA) has recently been reported to be one such inhibitor that also manifests immunogenic activity. Herein, we report preliminary structure-activity relationship (SAR) studies to determine the structural features that confer this unprecedented activity for an Hsp90 inhibitor. Our studies find that EnnA's branching moieties are necessary for its activity, but some structural modifications are tolerated.

Capsaicin binds the N-terminus of Hsp90, induces lysosomal degradation of Hsp70, and enhances the anti-tumor effects of 17-AAG (Tanespimycin).

Heat shock protein 90 (Hsp90) and its co-chaperones promote cancer, and targeting Hsp90 holds promise for cancer treatment. Most of the efforts to harness this potential have focused on targeting the Hsp90 N-terminus ATP binding site. Although newer-generation inhibitors have shown improved efficacy in aggressive cancers, induction of the cellular heat shock response (HSR) by these inhibitors is thought to limit their clinical efficacy. Therefore, Hsp90 inhibitors with novel mechanisms of action and that do not trigger the HSR would be advantageous. Here, we investigated the mechanism by which capsaicin inhibits Hsp90. Through mutagenesis, chemical modifications, and proteomic studies, we show that capsaicin binds to the N-terminus of Hsp90 and inhibits its ATPase activity. Consequently, capsaicin and its analogs inhibit Hsp90 ATPase-dependent progesterone receptor reconstitution in vitro. Capsaicin did not induce the HSR, instead, it promoted the degradation of Hsp70 through the lysosome-autophagy pathway. Remarkably, capsaicin did not induce degradation of the constitutively expressed cognate Hsc70, indicating selectivity for Hsp70. Combined treatments of capsaicin and the Hsp90 inhibitor 17-AAG improved the anti-tumor efficacy of 17-AAG in cell culture and tridimensional tumor spheroid growth assays using breast and prostate cancer models. Consistent with this, in silico docking studies revealed that capsaicin binding to the ATP binding site of Hsp90 was distinct from classical N-terminus Hsp90 inhibitors, indicating a novel mechanism of action. Collectively, these findings support the use of capsaicin as a chemical scaffold to develop novel Hsp90 N-terminus inhibitors as well as its ability to be a potential cancer co-therapeutic.

Enniatin A inhibits the chaperone Hsp90 and unleashes the immune system against triple-negative breast cancer.

Low response rates and immune-related adverse events limit the remarkable impact of cancer immunotherapy. To improve clinical outcomes, preclinical studies have shown that combining immunotherapies with N-terminal Hsp90 inhibitors resulted in improved efficacy, even though induction of an extensive heat shock response (HSR) and less than optimal dosing of these inhibitors limited their clinical efficacy as monotherapies. We discovered that the natural product Enniatin A (EnnA) targets Hsp90 and destabilizes its client oncoproteins without inducing an HSR. EnnA triggers immunogenic cell death in triple-negative breast cancer (TNBC) syngeneic mouse models and exhibits superior antitumor activity compared to Hsp90 N-terminal inhibitors. EnnA reprograms the tumor microenvironment (TME) to promote CD8+ T cell-dependent antitumor immunity by reducing PD-L1 levels and activating the chemokine receptor CX3CR1 pathway. These findings provide strong evidence for transforming the immunosuppressive TME into a more tumor-hostile milieu by engaging Hsp90 with therapeutic agents involving novel mechanisms of action.

Fluorinated thiazolidinol drives autophagic cell death in pancreatic cancer cells via AMPK activation and perturbation of critical sentinels of oncogenic signaling.

Pancreatic cancer is one of the most malignant cancers around the world. The co-occurrence of mutation in KRAS and p53 makes it highly aggressive, proliferative, metastatic, and resistant to apoptotic cell death. Therefore, there is a need to trigger an alternate mechanism of cancer cell death in apoptosis-resistant pancreatic cancer. Autophagic cell death could be an alternate viable option for treatment in such cases. Thus, the identification of small molecules as autophagy modulators with potent anticancer efficacy would be of great importance in pancreatic cancer. The present study investigates fluorinated thiazolidionol (FTZ) driven autophagy modulation, underlying mechanism, and regulation of critical sentinels of oncogenic signaling in pancreatic cancer cells. We identified that FTZ triggered autophagic cell death in pancreatic cancer cells, independent of apoptosis evidenced by an increase in cytoplasmic vacuoles formation, autophagy flux, LC3-II expression, and p62 degradation. Further, the crucial events of apoptosis i.e., Caspase-3 activation and PARP cleavage, were not observed, indicating the non-occurrence of apoptotic cell death. Moreover, FTZ was able to activate AMPK and suppress PI3k/Akt/mTOR as well as MEK/ERK, the key oncogenic signaling pathways in cancer cells. Furthermore, treatment with FTZ suppressed migration, invasion, and angiogenesis in pancreatic cancer cells. Studies in vivo revealed significant regression of tumors by FTZ in nude mice model. Overall, our study demonstrates that FTZ induces autophagic cell death in pancreatic cancer cells independent of apoptosis, which is accompanied by AMPK activation and suppression of critical sentinels of oncogenic signaling in pancreatic cancer cells.

Imidazo[2,1-b]thiazole-Coupled Natural Noscapine Derivatives as Anticancer Agents.

Noscapine, a phthalide isoquinoline alkaloid isolated from the opium poppy Papaver somniferum, is traditionally being used as an anticough drug. With a safe in vitro toxicological profile, noscapine and its analogues have been explored to show microtubule-regulating properties and anticancer activity against various mammalian cancer cell lines. Since then, our group and other research groups worldwide are working on developing new noscapinoids to tap its potential as the leading drug molecule. With our continuing efforts, we herein present synthesis and anticancer evaluation of a series of imidazothiazole-coupled noscapinoids 7a-o and 11a-o. Natural α-noscapine was N-demethylated to nornoscapine 4 and then reacted with 4-(chloromethyl) thiazole-2-amine. The resultant noscapinoid 5 was coupled with various bromomethyl ketones 10a-o to give N-imidazothiazolyl noscapinoids 7a-o in very good yields. Similarly, natural α-noscapine 1 was O-demethylated using sodium azide/sodium iodide, reacted with 4-(chloromethyl)thiazole-2-amine, and coupled with bromomethyl ketones 10a-o to result in O-imidazothiazolyl noscapinoids 11a-o. All the new analogues 7a-o and 11a-o were fully characterized by their NMR and mass spectral analysis. In vitro cytotoxicity assay was performed for compounds 5, 7a-o, 9, and 11a-o against four different cancer cell lines: HeLa (cervical), MIA PaCa-2 (pancreatic), SK-N-SH (neuroblastoma), and DU145 (prostate cancer). Among these conjugates, 5, 7a, 9, 11b, 11c, 11e, and 11o showed potent cytotoxicity with low IC50 values. Further, flow cytometry analysis revealed that MIA PaCa-2 cells treated with these compounds induced cell cycle G2/M-phase arrest. In addition, Western blot analysis revealed that the cells treated with these conjugates accumulate tubulin in the soluble fraction and also elevate cyclin-B1 protein expression levels. Moreover, the conjugates also increased the expression of caspase-3 and PARP levels which is indicative of apoptotic cell death. In silico molecular docking studies showed several noncovalent interactions like van der Waals and hydrogen-bonding with tubulin protein and with good binding energy. The results indicated that these noscapine analogues may serve as novel compounds that can possibly inhibit tubulin protein and can be considered for further optimization as a clinical candidate for treating pancreatic cancer.